Cowden Syndrome (CS) is an autosomal dominant inherited cancer syndrome where patients are at increased risk for developing cancer such as thyroid, breast, endometrial, and renal cancer. For ~25% of CS patients and ~5% of CS-like (CSL) patients, patients with many of the features of CS but do not meet strict criteria, germline mutation(s) have been identified in the PTEN gene. It is important for patient care including making a molecular diagnosis and preventative screening to understand the cause of CS/CSL in PTEN mutation negative patients. Recently, our lab identified germline hypermethylation of the gene KILLIN (KLLN) which is located 5' to PTEN and the two genes share a bidirectional promoter. 37% of CS/CSL patients were found to have KLLN hypermethylation which resulted in dramatically decreased KLLN expression, that is reversible by demethylating agents, and slightly increased PTEN expression. CS/CSL patients with KLLN hypermethylation had an increased risk, compared to PTEN mutation positive patients, for developing renal cell carcinoma and breast cancer. My proof of principal data demonstrates that there is KLLN promoter hypermethylation in a subset of CS/CSL patients with and without germline PTEN mutations. Furthermore we have shown KLLN mutations and methylation in patients with apparently sporadic breast cancer. Through this proposal, we will test the hypothesis, via genetic/epigenetic aims and a functional aim, that KLLN alterations lead to a CS/CSL phenotype with differences in prevalence of CS-related cancers and can particularly predispose women to breast cancer. The functional aim will focus on how KLLN alterations lead to heritable neoplasia through disruption of S-phase arrest and apoptosis. In aim 1 we will confirm KLLN hypermethylation in patients without PTEN mutations and analyze possible differences in prevalence of CS related cancers among patients. We will further analyze KLLN promoter hypermethylation in patients with pathogenic PTEN mutation(s) and how this could modify the CS cancer phenotype. Through aim 2, we will analyze KLLN exonic mutations and hypermethylation in patients with breast cancer. We will look for particular clinic- pathological features of breast cancer that are associated with KLLN alterations. For genetic evidence of KLLN as a tumor suppressor, we analyze the KLLN gene somatically for a second hit and look for absence of KLLN expression. For aim 3 we will examine the KLLN hypermethylation and mutations functionally. Knocking down KLLN will mimic hypermethylation and allow us to analyze the consequences of decreased KLLN on apoptosis and cell cycle regulation. KLLN exonic mutations will be analyzed for their detrimental effect on cellular regulation. We will then examine the consequences of having both a KLLN epimutation and a PTEN mutation. Overall through this grant, we will understand both the phenotypic consequences of a KLLN alterations as well as the altered cellular function due to these alterations, which will afford me rigorous training as a human cancer geneticist physician-scientist.